Tracking ligand-induced conformational changes through transmembrane sensory proteins remains a challenge in signal transduction research. Our study followed propagation of stimulus signals through a bacterial transmembrane chemoreceptor (Tsr) that controls a cytoplasmic signaling kinase (CheA). We marked relay elements in the ∼200-Å long cytoplasmic four-helix bundle signaling domain with amino acid replacements that locked Tsr in kinase-ON or -OFF output and characterized the mutant receptors with in vivo assays that monitored Tsr structure (crosslinking) and function (kinase control). We found that conformational changes emanating from the mutant lesions propagated bidirectionally throughout the Tsr signaling domain and did not dissipate appreciably with distance, implying conformational coupling between subdomains. These behaviors proved intrinsic to Tsr homodimers and independent of higher order signaling complexes. AlphaFold 3 atomic models of the mutant receptors exhibited structural changes that would likely affect local helix packing interactions at the mutant sites. One member of each ON-OFF mutant pair appeared to reduce packing stability, whereas the other enhanced packing stability. This analysis pinpointed two sites of structural logic inversion ("entropic switches") in the Tsr transmission path. Kinase-OFF lesions appeared to be stabilizing at the input and output segments of the cytoplasmic domain but destabilizing in the intervening signaling region that contains the modification sites for sensory adaptation. These findings support the notion of chemoreceptor signaling through opposed dynamic switches and provide new insights into the mechanism of kinase output control. This work also highlights the powerful interplay possible between cellular signaling readouts and AF3-generated models of mutant proteins.

The experimental challenges posed by integral membrane proteins hinder molecular understanding of transmembrane signaling mechanisms. Here, we exploited protein crosslinking assays in living cells to follow conformational and dynamic stimulus signals in Tsr, the Escherichia coli serine chemoreceptor. Tsr mediates serine chemotaxis by integrating transmembrane serine-binding inputs with adaptational modifications of a methylation helix bundle to regulate a signaling kinase at the cytoplasmic tip of the receptor molecule. We created cysteine replacements at Tsr residues adjacent to hydrophobic packing faces of the bundle helices and crosslinked them with a cell-permeable, bifunctional thiol-reagent. We identified an extensively crosslinked dynamic junction midway through the methylation helix bundle that seemed uniquely poised to respond to serine signals. We explored its role in mediating signaling shifts between different packing arrangements of the bundle helices by measuring crosslinking in receptor molecules with apposed pairs of cysteine reporters in each subunit and assessing their signaling behaviors with an in vivo kinase assay. In the absence of serine, the bundle helices evinced compact kinase-ON packing arrangements; in the presence of serine, the dynamic junction destabilized adjacent bundle segments and shifted the bundle to an expanded, less stable kinase-OFF helix-packing arrangement. AlphaFold models of kinase-active Tsr showed a prominent bulge and kink at the dynamic junction that might antagonize stable structure at the receptor tip. Serine stimuli might inhibit kinase activity by shifting the bundle to a less stably-packed conformation that relaxes structural strain at the receptor tip, thereby allowing it to stabilize and freeze kinase activity.

The chemotaxis machinery of Escherichia coli has served as a model for exploring the molecular signaling mechanisms of transmembrane chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). Yet, fundamental questions about signal transmission through MCP molecules remain unanswered. Our work with the E. coli serine chemoreceptor Tsr has developed in vivo reporters that distinguish kinase-OFF and kinase-ON structures in the cytoplasmic methylation helix (MH) cap, which receives stimulus signals from an adjoining, membrane-proximal histidine kinase, adenylyl cyclases, MCPs, and phosphatases (HAMP) domain. The cytoplasmic helices of the Tsr homodimer interact mainly through packing interactions of hydrophobic residues at a and d heptad positions. We investigated the in vivo crosslinking properties of Tsr molecules bearing cysteine replacements at functionally tolerant g heptad positions in the N-terminal and C-terminal cap helices. Upon treatment of cells with bismaleimidoethane (BMOE), a bifunctional thiol-reagent, Tsr-G273C/Q504C readily formed a doubly crosslinked product in the presence of serine but not in its absence. Moreover, a serine stimulus combined with BMOE treatment during in vivo F_orster resonance energy transfer-based kinase assays locked Tsr-G273C/Q504C in kinase-OFF output. An OFF-shifting lesion in MH1 (D269P) promoted the formation of the doubly crosslinked species in the absence of serine, whereas an ON-shifting lesion (G268P) suppressed the formation of the doubly crosslinked species. Tsr-G273C/Q504C also showed output-dependent crosslinking patterns in combination with ON-shifting and OFF-shifting adaptational modifications. Our results are consistent with a helix breathing-axial rotation-bundle repacking signaling mechanism and imply that in vivo crosslinking tools could serve to probe helix-packing transitions and their output consequences in other regions of the receptor molecule.

Staphylococcus aureus (S. aureus) causes a range of diseases ranging from superficial skin and soft-tissue infections to invasive and life-threatening conditions (Klevens et al., 2007; Kobayashi et al., 2015). S. aureus utilizes the Sae sensory system to adapt to neutrophil challenge. Although the roles of the SaeR response regulator and its cognate sensor kinase SaeS have been demonstrated to be critical for surviving neutrophil interaction and for causing infection, the roles for the accessory proteins SaeP and SaeQ remain incompletely defined. To characterize the functional role of these proteins during innate immune interaction, we generated isogenic deletion mutants lacking these accessory genes in USA300 (USA300ΔsaeP and USA300ΔsaeQ). S. aureus survival was increased following phagocytosis of USA300ΔsaeP compared to USA300 by neutrophils. Additionally, secreted extracellular proteins produced by USA300ΔsaeP cells caused significantly more plasma membrane damage to human neutrophils than extracellular proteins produced by USA300 cells. Deletion of saeQ resulted in a similar phenotype, but effects did not reach significance during neutrophil interaction. The enhanced cytotoxicity of USA300ΔsaeP cells toward human neutrophils correlated with an increased expression of bi-component leukocidins known to target these immune cells. A saeP and saeQ double mutant (USA300ΔsaePQ) showed a significant increase in survival following neutrophil phagocytosis that was comparable to the USA300ΔsaeP single mutant and increased the virulence of USA300 during murine bacteremia. These data provide evidence that SaeP modulates the Sae-mediated response of S. aureus against human neutrophils and suggest that saeP and saeQ together impact pathogenesis in vivo.

We developed a new approach that couples Southwestern blotting and mass spectrometry to discover proteins that bind extracellular DNA (eDNA) in bacterial biofilms. Using Staphylococcus aureus as a model pathogen, we identified proteins with known DNA-binding activity and uncovered a series of lipoproteins with previously unrecognized DNA-binding activity. We demonstrated that expression of these lipoproteins results in an eDNA-dependent biofilm enhancement. Additionally, we found that while deletion of lipoproteins had a minimal impact on biofilm accumulation, these lipoprotein mutations increased biofilm porosity, suggesting that lipoproteins and their associated interactions contribute to biofilm structure. For one of the lipoproteins, SaeP, we showed that the biofilm phenotype requires the lipoprotein to be anchored to the outside of the cellular membrane, and we further showed that increased SaeP expression correlates with more retention of high-molecular-weight DNA on the bacterial cell surface. SaeP is a known auxiliary protein of the SaeRS system, and we also demonstrated that the levels of SaeP correlate with nuclease production, which can further impact biofilm development. It has been reported that S. aureus biofilms are stabilized by positively charged cytoplasmic proteins that are released into the extracellular environment, where they make favorable electrostatic interactions with the negatively charged cell surface and eDNA. In this work we extend this electrostatic net model to include secreted eDNA-binding proteins and membrane-attached lipoproteins that can function as anchor points between eDNA in the biofilm matrix and the bacterial cell surface.IMPORTANCE Many bacteria are capable of forming biofilms encased in a matrix of self-produced extracellular polymeric substances (EPS) that protects them from chemotherapies and the host defenses. As a result of these inherent resistance mechanisms, bacterial biofilms are extremely difficult to eradicate and are associated with chronic wounds, orthopedic and surgical wound infections, and invasive infections, such as infective endocarditis and osteomyelitis. It is therefore important to understand the nature of the interactions between the bacterial cell surface and EPS that stabilize biofilms. Extracellular DNA (eDNA) has been recognized as an EPS constituent for many bacterial species and has been shown to be important in promoting biofilm formation. Using Staphylococcus aureus biofilms, we show that membrane-attached lipoproteins can interact with the eDNA in the biofilm matrix and promote biofilm formation, which suggests that lipoproteins are potential targets for novel therapies aimed at disrupting bacterial biofilms.

Environmental awareness is an essential attribute for all organisms. The chemotaxis system of Escherichia coli provides a powerful experimental model for the investigation of stimulus detection and signaling mechanisms at the molecular level. These bacteria sense chemical gradients with transmembrane proteins [methyl-accepting chemotaxis proteins (MCPs)] that have an extracellular ligand-binding domain and intracellular histidine kinases, adenylate cyclases, methyl-accepting proteins, and phosphatases (HAMP) and signaling domains that govern locomotor behavior. HAMP domains are versatile input-output elements that operate in a variety of bacterial signaling proteins, including the sensor kinases of two-component regulatory systems. The MCP HAMP domain receives stimulus information and in turn modulates output signaling activity. This study describes mutants of the Escherichia coli serine chemoreceptor, Tsr, that identify a heptad-repeat structural motif (LLF) at the membrane-proximal end of the receptor signaling domain that is critical for HAMP output control. The homodimeric Tsr signaling domain is an extended, antiparallel, four-helix bundle that controls the activity of an associated kinase. The N terminus of each subunit adjoins the HAMP domain; the LLF residues lie at the C terminus of the methylation-helix bundle. We found, by using in vivo Förster resonance energy transfer kinase assays, that most amino acid replacements at any of the LLF residues abrogate chemotactic responses to serine and lock Tsr output in a kinase-active state, impervious to HAMP-mediated down-regulation. We present evidence that the LLF residues may function like a leucine zipper to promote stable association of the C-terminal signaling helices, thereby creating a metastable helix-packing platform for the N-terminal signaling helices that facilitates conformational control by the HAMP domains in MCP-family chemoreceptors.

Significance Staphylococcus aureus is a ubiquitous pathogen that causes disease in a variety of tissues. Our studies provide insight into how this pathogen uses the SaeS sensor kinase to recognize innate immunity signals and induce a transcriptional response tailored for its environment. Our results demonstrate that the specificity of these responses is determined by individual amino acids predicted to be in an extracellular domain. These amino acids include aromatic anchors and a methionine residue essential for activation of target gene transcription and virulence. Our findings provide a putative mechanism for the ability of bacterial sensory systems to integrate and diversify the responses to host stimuli. They underscore the exquisite nature of bacterial signaling and the complexity of host-pathogen interactions.

ABSTRACT Several prominent bacterial pathogens secrete nuclease (Nuc) enzymes that have an important role in combating the host immune response. Early studies of Staphylococcus aureus Nuc attributed its regulation to the agr quorum-sensing system. However, recent microarray data have indicated that nuc is under the control of the SaeRS two-component system, which is a major regulator of S. aureus virulence determinants. Here we report that the nuc gene is directly controlled by the SaeRS two-component system through reporter fusion, immunoblotting, Nuc activity measurements, promoter mapping, and binding studies, and additionally, we were unable identify a notable regulatory link to the agr system. The observed SaeRS-dependent regulation was conserved across a wide spectrum of representative S. aureus isolates. Moreover, with community-associated methicillin-resistant S. aureus (CA MRSA) in a mouse model of peritonitis, we observed in vivo expression of Nuc activity in an SaeRS-dependent manner and determined that Nuc is a virulence factor that is important for in vivo survival, confirming the enzyme's role as a contributor to invasive disease. Finally, natural polymorphisms were identified in the SaeRS proteins, one of which was linked to Nuc regulation in a CA MRSA USA300 endocarditis isolate. Altogether, our findings demonstrate that Nuc is an important S. aureus virulence factor and part of the SaeRS regulon.

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTPeptide Signaling in the StaphylococciMatthew Thoendel, Jeffrey S. Kavanaugh, Caralyn E. Flack, and Alexander R. Horswill*View Author Information Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, United States* To whom correspondence should be addressed. Phone: 319 335 7783. Fax: 319 335 8228. E-mail: [email protected]. Address: 540F EMRB, University of Iowa, Iowa City, Iowa 52242.Cite this: Chem. Rev. 2011, 111, 1, 117–151Publication Date (Web):December 21, 2010Publication History Received29 October 2010Published online21 December 2010Published inissue 12 January 2011https://pubs.acs.org/doi/10.1021/cr100370nhttps://doi.org/10.1021/cr100370nreview-articleACS PublicationsCopyright © 2010 American Chemical SocietyRequest reuse permissionsArticle Views4521Altmetric-Citations318LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Biofilms,Genetics,Infectious diseases,Monomers,Peptides and proteins Get e-Alerts

In the postantibiotic era, available treatment options for severe bacterial infections caused by methicillin-resistant Staphylococcus aureus have become limited. Therefore, new and innovative approaches are needed to combat such life-threatening infections. Virulence factor expression in S. aureus is regulated in a cell density-dependent manner using “quorum sensing,” which involves generation and secretion of autoinducing peptides (AIPs) into the surrounding environment to activate a bacterial sensor kinase at a particular threshold concentration. Mouse monoclonal antibody AP4-24H11 was shown previously to blunt quorum sensing-mediated changes in gene expression in vitro and protect mice from a lethal dose of S. aureus by sequestering the AIP signal. We have elucidated the crystal structure of the AP4-24H11 Fab in complex with AIP-4 at 2.5 Å resolution to determine its mechanism of ligand recognition. A key GluH95 provides much of the binding specificity through formation of hydrogen bonds with each of the four amide nitrogens in the AIP-4 macrocyclic ring. Importantly, these structural data give clues as to the interactions between the cognate staphylococcal AIP receptors AgrC and the AIPs, as AP4-24H11·AIP-4 binding recapitulates features that have been proposed for AgrC-AIP recognition. Additionally, these structural insights may enable the engineering of AIP cross-reactive antibodies or quorum quenching vaccines for use in active or passive immunotherapy for prevention or treatment of S. aureus infections.

The existence of flux ratio anomalies between fold and cusp images in galaxy-scale strong-lens systems has led to an interpretation based on the presence of a high mass fraction of cold dark matter (CDM) substructures around galaxies, as predicted by numerical N-body simulations. These substructures can cause large perturbations of the image magnifications, leading to changes in the image flux ratios. The flux ratio anomaly is particularly evident in the radio-loud quadruple gravitational lens system CLASS B2045+265. In this paper, new high-resolution radio, optical and infrared imaging of B2045+265 is presented which sheds more light on this anomaly and its possible causes. First, deep Very Long Baseline Array observations show very compact images, possibly with a hint of a jet, but with no evidence for differential scattering or scatter broadening. Hence, the flux ratio anomaly is unlikely to be caused by refractive scattering in either the Milky Way or the lens galaxy. Secondly, optical and infrared observations with the Hubble Space Telescope and through adaptive optics imaging with the W. M. Keck Telescope, show a previously undiscovered object - interpreted as a (tidally disrupted) dwarf satellite based on its colours and slight extension - between the main lens galaxy and the three anomalous flux ratio images. Thirdly, colour variations in the early-type lens galaxy indicate recent star formation, possibly the result of secondary infall of gas-rich satellites. A population of such galaxies around the lens system could explain the previously discovered strong [O II] emission. However, spiral structure and/or normal star formation in the lens galaxy cannot be excluded. In light of these new data, we propose a lens model for the system, including the observed dwarf satellite, which reproduces all positional and flux ratio constraints, without the need for additional CDM substructure. Although the model is peculiar in that the dwarf galaxy must be highly flattened, the model is very similar to recently proposed mass models based on high-order multipole expansions.