Bacitracin sensing and resistance in Staphylococcus aureus

Bacterial two-component systems (TCSs) have been demonstrated to be associated with not only the expression of virulence factors, but also the susceptibility to antibacterial agents. In Staphylococcus aureus, 16 types of TCSs have been identified. We previously found that the inactivation of one uncharacterized TCS (designated as BceRS, MW gene ID: MW2545-2544) resulted in an increase in susceptibility to bacitracin. In this study, we focused on this TCS and tried to identify the TCS-controlled factors affecting the susceptibility to bacitracin. We found that two ABC transporters were associated with the susceptibility to bacitracin. One transporter designated as BceAB (MW2543-2542) is downstream of this TCS, while another (formerly designated as VraDE: MW2620-2621) is separate from this TCS. Both transporters showed homology with several bacitracin-resistance factors in Gram-positive bacteria. Inactivation of each of these two transporters increased the susceptibility to bacitracin. Expressions of these transporters were significantly increased by the addition of bacitracin, while this induction was not observed in the TCS-inactivated mutant. These results indicate that this TCS senses bacitracin, and also positively regulates the expression of two ABC transporters.     

The yydFGHIJ operon of Bacillus subtilis encodes a peptide that induces the LiaRS two-component system

The Bacillus subtilis LiaRS two-component system (TCS) responds to perturbations of the cell envelope induced by lipid II-interacting antibiotics, such as vancomycin, ramoplanin, nisin, and bacitracin. Here, we characterize Tn7-generated mutations that induce the liaRS TCS. In addition to insertions in liaF, a known negative regulator of the LiaRS TCS, we identified two disruptions in the last two genes of the yydFGHIJ operon. This operon is predicted to encode a 49-amino-acid peptide (YydF), a modification enzyme (YydG), a membrane-embedded protease (YydH), and an ATP-binding cassette (ABC) transporter (YydIJ). Genome sequence comparisons suggest that the yydFGHIJ operon may have been acquired by horizontal transfer. Inactivation of the YydIJ transporter resulted in increased expression from the LiaR-dependent P_liaI promoter only in the presence of the yydFGH genes. Cells harboring the complete yydFGHIJ operon induced LiaR activity in cocultured cells lacking either this transporter or the complete operon. These results suggest that this operon is involved in the synthesis and export of a modified peptide (YydF*) that elicits cell envelope stress sensed by the LiaRS TCS.     

Anatomy of the bacitracin resistance network in Bacillus subtilis

Protection against antimicrobial peptides (AMPs) often involves the parallel production of multiple, well‐characterized resistance determinants. So far, little is known about how these resistance modules interact and how they jointly protect the cell. Here, we studied the interdependence between different layers of the envelope stress response of Bacillus subtilis when challenged with the lipid II cycle‐inhibiting AMP bacitracin. The underlying regulatory network orchestrates the production of the ABC transporter BceAB, the UPP phosphatase BcrC and the phage‐shock proteins LiaIH. Our systems‐level analysis reveals a clear hierarchy, allowing us to discriminate between primary (BceAB) and secondary (BcrC and LiaIH) layers of bacitracin resistance. Deleting the primary layer provokes an enhanced induction of the secondary layer to partially compensate for this loss. This study reveals a direct role of LiaIH in bacitracin resistance, provides novel insights into the feedback regulation of the Lia system, and demonstrates a pivotal role of BcrC in maintaining cell wall homeostasis. The compensatory regulation within the bacitracin network can also explain how gene expression noise propagates between resistance layers. We suggest that this active redundancy in the bacitracin resistance network of B. subtilis is a general principle to be found in many bacterial antibiotic resistance networks.     

A Sensory Complex Consisting of an ATP-binding Cassette Transporter and a Two-component Regulatory System Controls Bacitracin Resistance in Bacillus subtilis

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems.     

Coevolution of ABC transporters and two-component regulatory systems as resistance modules against antimicrobial peptides in Firmicutes Bacteria

In Firmicutes bacteria, ATP-binding cassette (ABC) transporters have been recognized as important resistance determinants against antimicrobial peptides. Together with neighboring two-component systems (TCSs), which regulate their expression, they form specific detoxification modules. Both the transport permease and sensor kinase components show unusual domain architecture: the permeases contain a large extracellular domain, while the sensor kinases lack an obvious input domain. One of the best-characterized examples is the bacitracin resistance module BceRS-BceAB of Bacillus subtilis. Strikingly, in this system, the ABC transporter and TCS have an absolute mutual requirement for each other in both sensing of and resistance to bacitracin, suggesting a novel mode of signal transduction in which the transporter constitutes the actual sensor. We identified over 250 such BceAB-like ABC transporters in the current databases. They occurred almost exclusively in Firmicutes bacteria, and 80% of the transporters were associated with a BceRS-like TCS. Phylogenetic analyses of the permease and sensor kinase components revealed a tight evolutionary correlation. Our findings suggest a direct regulatory interaction between the ABC transporters and TCSs, mediating communication between both components. Based on their observed coclustering and conservation of response regulator binding sites, we could identify putative corresponding two-component systems for transporters lacking a regulatory system in their immediate neighborhood. Taken together, our results show that these types of ABC transporters and TCSs have coevolved to form self-sufficient detoxification modules against antimicrobial peptides, widely distributed among Firmicutes bacteria.     

Identification of a two-component regulatory system involved in antimicrobial peptide resistance in Streptococcus pneumoniae

Two-component regulatory systems (TCS) are among the most widespread mechanisms that bacteria use to sense and respond to environmental changes. In the human pathogen Streptococcus pneumoniae, a total of 13 TCS have been identified and many of them have been linked to pathogenicity. Notably, TCS01 strongly contributes to pneumococcal virulence in several infection models. However, it remains one of the least studied TCS in pneumococci and its functional role is still unclear. In this study, we demonstrate that TCS01 cooperates with a BceAB-type ABC transporter to sense and induce resistance to structurally-unrelated antimicrobial peptides of bacterial origin that all target undecaprenyl-pyrophosphate or lipid II, which are essential precursors of cell wall biosynthesis. Even though tcs01 and bceAB genes do not locate in the same gene cluster, disruption of either of them equally sensitized the bacterium to the same set of antimicrobial peptides. We show that the key function of TCS01 is to upregulate the expression of the transporter, while the latter appears the main actor in resistance. Electrophoretic mobility shift assays further demonstrated that the response regulator of TCS01 binds to the promoter region of the bceAB genes, implying a direct control of these genes. The BceAB transporter was overexpressed and purified from E. coli. After reconstitution in liposomes, it displayed substantial ATPase and GTPase activities that were stimulated by antimicrobial peptides to which it confers resistance to, revealing new functional features of a BceAB-type transporter. Altogether, this inducible defense mechanism likely contributes to the survival of the opportunistic microorganism in the human host, in which competition among commensal microorganisms is a key determinant for effective host colonization and invasive path.     

Accumulation of heptaprenyl diphosphate sensitizes Bacillus subtilis to bacitracin: implications for the mechanism of resistance mediated by the BceAB transporter

Heptaprenyl diphosphate (C₃₅‐PP) is an isoprenoid intermediate in the synthesis of both menaquinone and the sesquarterpenoids. We demonstrate that inactivation of ytpB, encoding a C₃₅‐PP utilizing enzyme required for sesquarterpenoid synthesis, leads to an increased sensitivity to bacitracin, an antibiotic that binds undecaprenyl pyrophosphate (C₅₅‐PP), a key intermediate in cell wall synthesis. Genetic studies indicate that bacitracin sensitivity is due to accumulation of C₃₅‐PP, rather than the absence of sesquarterpenoids. Sensitivity is accentuated in a ytpB menA double mutant, lacking both known C₃₅‐PP consuming enzymes, and in a ytpB strain overexpressing the HepST enzyme that synthesizes C₃₅‐PP. Conversely, sensitivity in the ytpB background is suppressed by mutation of hepT or by supplementation with 1,4‐dihydroxy‐2‐naphthoate, a co‐substrate with C₃₅‐PP for MenA. Bacitracin sensitivity results from impairment of the BceAB and BcrC resistance mechanisms by C₃₅‐PP: in a bceAB bcrC double mutant disruption of ytpB no longer increases bacitracin sensitivity. These results suggest that C₃₅‐PP inhibits both BcrC (a C₅₅‐PP phosphatase) and BceAB (an ABC transporter that confers bacitracin resistance). These findings lead to a model in which BceAB protects against bacitracin by transfer of the target, C₅₅‐PP, rather than the antibiotic across the membrane.     

YtsCD and YwoA, two independent systems that confer bacitracin resistance to Bacillus subtilis

The Bacillus subtilis yts, yxd and yvc gene clusters encode a putative ABC transporter and a functionally coupled two-component system. When tested for their sensitivity towards a series of antibiotics, null yts mutants were found to be sensitive to bacitracin. Real-time polymerase chain reaction (PCR) experiments demonstrated that the presence of bacitracin in the growth medium strongly stimulates the expression of the ytsCD genes encoding the ABC transporter and that this stimulation strictly depends on the YtsA response regulator. The ywoA gene encodes a protein known to confer some resistance to bacitracin on the bacterium. When it was mutated in a null yts background, the ywoA yts double mutant was found to be five times more sensitive than the yts one. We propose that (i) the YtsCD ABC transporter exports the bacitracin; (ii) YwoA, the protein that contains an acidPPc (PAP2 or PgpB) domain, is not part of an ABC transporter but competes with bacitracin for the dephosphorylation of the C55-isoprenyl pyrophosphate (IPP); (iii) the two resistance mechanisms are independent and complementary.     

Immediate and Heterogeneous Response of the LiaFSR Two-Component System of Bacillus subtilis to the Peptide Antibiotic Bacitracin

Background

Two-component signal transduction systems are one means of bacteria to respond to external stimuli. The LiaFSR two-component system of Bacillus subtilis consists of a regular two-component system LiaRS comprising the core Histidine Kinase (HK) LiaS and the Response Regulator (RR) LiaR and additionally the accessory protein LiaF, which acts as a negative regulator of LiaRS-dependent signal transduction. The complete LiaFSR system was shown to respond to various peptide antibiotics interfering with cell wall biosynthesis, including bacitracin.

Methodology and Principal Findings

Here we study the response of the LiaFSR system to various concentrations of the peptide antibiotic bacitracin. Using quantitative fluorescence microscopy, we performed a whole population study analyzed on the single cell level. We investigated switching from the non-induced ‘OFF’ state into the bacitracin-induced ‘ON’ state by monitoring gene expression of a fluorescent reporter from the RR-regulated liaI promoter. We found that switching into the ‘ON’ state occurred within less than 20 min in a well-defined switching window, independent of the bacitracin concentration. The switching rate and the basal expression rate decreased at low bacitracin concentrations, establishing clear heterogeneity 60 min after bacitracin induction. Finally, we performed time-lapse microscopy of single cells confirming the quantitative response as obtained in the whole population analysis for high bacitracin concentrations.

Conclusion

The LiaFSR system exhibits an immediate, heterogeneous and graded response to the inducer bacitracin in the exponential growth phase.     

The BceRS two-component regulatory system induces expression of the bacitracin transporter,BceAB, in Bacillus subtilis

BceA and bceB encode a nucleotide-binding domain (NBD) and membrane-spanning domain (MSD) subunit, respectively, of an ATP-binding cassette (ABC) transporter in Bacillus subtilis. Disruption of these genes resulted in hypersensitivity to bacitracin, a peptide antibiotic that is non-ribosomally synthesized in some strains of Bacillus. Northern hybridization analyses showed that expression of the bceAB operon is induced by bacitracin present in the growth medium. The bceRS genes encoding a two-component regulatory system are located immediately upstream of bceAB. Deletion analyses of the bceAB promoter together with DNase I footprinting experiments revealed that a sensor kinase, BceS, responds to extracellular bacitracin either directly or indirectly and transmits a signal to a cognate response regulator, BceR. The regulator binds directly to the upstream region of the bceAB promoter and upregulates the expression of bceAB genes. The bcrC gene product is additionally involved in bacitracin resistance. The expression of bcrC is dependent on the ECF sigma factors, sigmaM and sigmaX, but not on the BceRS two-component system. In view of these results, possible roles of BceA, BceB and BcrC in bacitracin resistance of B. subtilis 168 are discussed.     

Two-Component System Cross-Regulation Integrates Bacillus anthracis Response to Heme and Cell Envelope Stress

Two-component signaling systems (TCSs) are one of the mechanisms that bacteria employ to sense and adapt to changes in the environment. A prototypical TCS functions as a phosphorelay from a membrane-bound sensor histidine kinase (HK) to a cytoplasmic response regulator (RR) that controls target gene expression. Despite significant homology in the signaling domains of HKs and RRs, TCSs are thought to typically function as linear systems with little to no cross-talk between non-cognate HK-RR pairs. Here we have identified several cell envelope acting compounds that stimulate a previously uncharacterized Bacillus anthracis TCS. Furthermore, this TCS cross-signals with the heme sensing TCS HssRS; therefore, we have named it HssRS interfacing TCS (HitRS). HssRS reciprocates cross-talk to HitRS, suggesting a link between heme toxicity and cell envelope stress. The signaling between HssRS and HitRS occurs in the parental B. anthracis strain; therefore, we classify HssRS-HitRS interactions as cross-regulation. Cross-talk between HssRS and HitRS occurs at both HK-RR and post-RR signaling junctions. Finally, HitRS also regulates a previously unstudied ABC transporter implicating this transporter in the response to cell envelope stress. This chemical biology approach to probing TCS signaling provides a new model for understanding how bacterial signaling networks are integrated to enable adaptation to complex environments such as those encountered during colonization of the vertebrate host.     

Heat-shock and general stress response in Bacillus subtilis

The induction of stress proteins is an important component of the adaptional network of a non-growing cell of Bacillus subtilis . A diverse range of stresses such as heat shock, salt stress, ethanol, starvation for oxygen or nutrients etc. induce the same set of proteins, called general stress proteins. Although the adaptive functions of these proteins are largely unknown, they are proposed to provide general and rather non-specific protection of the cell under these adverse conditions. In addition to these non-specific general stress proteins, all extracellular signals induce a set of specific stress proteins that may confer specific protection against a particular stress factor. In B. subtilis at least three different classes of heat-inducible genes can be defined by their common regulatory characteristics: Class I genes, as exemplified by the dnaK and groE operons, are most efficiently induced by heat stress. Their expression involves a σ^A-dependent promoter, an inverted repeat (called the CIRCE element) highly conserved among eubacteria, and probably a repressor interacting with the CIRCE element. The majority of general stress genes (class II, more than 40) are induced at σ^B-dependent promoters by different growth-inhibiting conditions. The activation of σ^B by stress or starvation is the crucial event in the induction of this large stress regulon. Only a few genes, including lon clpC clpP , and ftsH, can respond to different stress factors independently of σ^B or CIRCE (class III). Stress induction of these genes occurs at promoters presumably recognized by σ^A and probably involves additional regulatory elements which remain to be defined.