Antimicrobial Peptide Recognition Motif of the Substrate Binding Protein SapA from Nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen associated with respiratory diseases, including otitis media and exacerbations of chronic obstructive pulmonary disease. NTHi exhibits resistance to killing by host antimicrobial peptides (AMPs) mediated by SapA, the substrate binding protein of the sensitivity to antimicrobial peptides (Sap) transporter. However, the specific mechanisms by which SapA selectively binds various AMPs such as defensins and cathelicidin are unknown. In this study, we report mutational analyses of both defensin AMPs and the SapA binding pocket to define the specificity of AMP recognition. Bactericidal assays revealed that NTHi lacking SapA are more susceptible to human beta defensins and LL-37, while remaining highly resistant to a human alpha defensin. In contrast to homologues, our research underscores the distinct specificity of NTHi SapA, which selectively recognizes and binds to peptides containing the charged-hydrophobic motif PKE and RRY. These findings provide valuable insight into the divergence of SapA among bacterial species and NTHi SapA's ability to selectively interact with specific AMPs to mediate resistance.

Microevolution in response to transient heme-iron restriction enhances intracellular bacterial community development and persistence.

Bacterial pathogens must sense, respond and adapt to a myriad of dynamic microenvironmental stressors to survive. Adaptation is key for colonization and long-term ability to endure fluctuations in nutrient availability and inflammatory processes. We hypothesize that strains adapted to survive nutrient deprivation are more adept for colonization and establishment of chronic infection. In this study, we detected microevolution in response to transient nutrient limitation through mutation of icc. The mutation results in decreased 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in nontypeable Haemophilus influenzae (NTHI). In a preclinical model of NTHI-induced otitis media (OM), we observed a significant decrease in the recovery of effusion from ears infected with the icc mutant strain. Clinically, resolution of OM coincides with the clearance of middle ear fluid. In contrast to this clinical paradigm, we observed that the icc mutant strain formed significantly more intracellular bacterial communities (IBCs) than the parental strain early during experimental OM. Although the number of IBCs formed by the parental strain was low at early stages of OM, we observed a significant increase at later stages that coincided with absence of recoverable effusion, suggesting the presence of a mucosal reservoir following resolution of clinical disease. These data provide the first insight into NTHI microevolution during nutritional limitation and provide the first demonstration of IBCs in a preclinical model of chronic OM.

Sensitivity of Magnetic Anisotropy in the Solid State for Lanthanide Complexes with Small Crystal Field Splitting.

Knowledge of the crystal structure of a monometallic inorganic molecule is often sufficient to calculate its electronic structure and interpret its magnetic properties. Here we show that for a series of nine-coordinate lanthanide complexes based on the 1,4,7-tris[(6-carboxypyridin-2-yl)methyl]-1,4,7-triazacyclononane ligand, the electronic structure is hypersensitive to geometric structure and to the presence of noncoordinated lattice solvent, which renders the magnetic and spectroscopic properties very difficult to interpret. We explore possible explanations for the peculiar electron paramagnetic resonance (EPR) spectra and conclude that a number of entangled factors are at play across the samples. Hence, great care should be taken in the interpretation of EPR spectra for systems with small magnetic anisotropy, even when the molecular structure is known.

Unravelling the Complexities of Pseudocontact Shift Analysis in Lanthanide Coordination Complexes of Differing Symmetry.

In two closely related series of eight-coordinate lanthanide complexes, a switch in the sign of the dominant ligand field parameter and striking variations in the sign, amplitude and orientation of the main component of the magnetic susceptibility tensor as the Ln3+ ion is permuted conspire to mask modest changes in NMR paramagnetic shifts, but are evident in Yb EPR and Eu emission spectra.

Selective substrate uptake: The role of ATP-binding cassette (ABC) importers in pathogenesis.

The uptake of nutrients, including metals, amino acids and peptides are required for many biological processes. Pathogenic bacteria scavenge these essential nutrients from microenvironments to survive within the host. Pathogens must utilize a myriad of mechanisms to acquire these essential nutrients from the host while mediating the effects of toxicity. Bacteria utilize several transport proteins, including ATP-binding cassette (ABC) transporters to import and expel substrates. ABC transporters, conserved across all organisms, are powered by the energy from ATP to move substrates across cellular membranes. In this review, we will focus on nutrient uptake, the role of ABC importers at the host-pathogen interface, and explore emerging therapies to combat pathogenesis. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

A novel streptococcal cell-cell communication peptide promotes pneumococcal virulence and biofilm formation.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen. It is a common colonizer of the human respiratory track, where it utilizes cell-cell communication systems to coordinate population-level behaviors. We reasoned that secreted peptides that are highly expressed during infection are pivotal for virulence. Thus, we used in silico pattern searches to define a pneumococcal secretome and analyzed the transcriptome of the clinically important PMEN1 lineage to identify which peptide-encoding genes are highly expressed in vivo. In this study, we characterized virulence peptide 1 (vp1), a highly expressed Gly-Gly peptide-encoding gene in chinchilla middle ear effusions. The vp1 gene is widely distributed across pneumococcus as well as encoded in related species. Studies in the chinchilla model of middle ear infection demonstrated that VP1 is a virulence determinant. The vp1 gene is positively regulated by a transcription factor from the Rgg family and its cognate SHP (short hydrophobic peptide). In vitro data indicated that VP1 promotes increased thickness and biomass for biofilms grown on chinchilla middle ear epithelial cells. Furthermore, the wild-type biofilm is restored with the exogenous addition of synthetic VP1. We conclude that VP1 is a novel streptococcal regulatory peptide that controls biofilm development and pneumococcal pathogenesis.

Aggregation of Rare Earth Coordination Complexes in Solution Studied by Paramagnetic and DOSY NMR.

The degree of aggregation of neutral, 9-coordinate rare earth coordination complexes has been shown to affect their ligand field, as revealed by diffusion-ordered NMR spectroscopy (DOSY-NMR) measurements on Y(III) complexes, paramagnetic NMR analyses of Yb and Tb analogues and emission spectral studies with the EuIII systems. In non-polar media a lipophilic tris-isopropyl complex, [Ln.L2 ] tends to aggregate in chloroform and dichloromethane giving rise to oligomers, whereas in acetic and trifluoroacetic acid the more polar parent complex, [Ln.L1 ], also aggregates, profoundly affecting the pseudocontact shift and the form of the Eu emission spectrum. Such behaviour has important implications in the design of responsive spectral probes.

Lanthanide-induced relaxation anisotropy.

Lanthanide ions accelerate nuclear spin relaxation by two primary mechanisms: dipolar and Curie. Both are commonly assumed to depend on the length of the lanthanide-nucleus vector, but not on its direction. Here we show experimentally that this is wrong - careful proton relaxation data analysis in a series of isostructural lanthanide complexes (Ln = Tb, Dy, Ho, Er, Tm, Yb) reveals angular dependence in both Curie and dipolar relaxation. The reasons are: (a) that magnetic susceptibility anisotropy can be of the same order of magnitude as the isotropic part (contradicting the unstated assumption in Guéron's theory of the Curie relaxation process), and (b) that zero-field splitting can be much stronger than the electron Zeeman interaction (Bloembergen's original theory of the lanthanide-induced dipolar relaxation process makes the opposite assumption). These factors go beyond the well researched cross-correlation effects; they alter the relaxation theory treatment and make strong angular dependencies appear in the nuclear spin relaxation rates. Those dependencies are impossible to ignore - this is now demonstrated both theoretically and experimentally, and suggests that a major revision is needed of the way lanthanide-induced relaxation data are used in structural biology.

Comprehensive Proteomic and Metabolomic Signatures of Nontypeable Haemophilus influenzae-Induced Acute Otitis Media Reveal Bacterial Aerobic Respiration in an Immunosuppressed Environment.

A thorough understanding of the molecular details of the interactions between bacteria and host are critical to ultimately prevent disease. Recent technological advances allow simultaneous analysis of host and bacterial protein and metabolic profiles from a single small tissue sample to provide insight into pathogenesis. We used the chinchilla model of human otitis media to determine, for the first time, the most expansive delineation of global changes in protein and metabolite profiles during an experimentally induced disease. After 48 h of infection with nontypeable Haemophilus influenzae, middle ear tissue lysates were analyzed by high-resolution quantitative two-dimensional liquid chromatography-tandem mass spectrometry. Dynamic changes in 105 chinchilla proteins and 66 metabolites define the early proteomic and metabolomic signature of otitis media. Our studies indicate that establishment of disease coincides with actin morphogenesis, suppression of inflammatory mediators, and bacterial aerobic respiration. We validated the observed increase in the actin-remodeling complex, Arp2/3, and experimentally showed a role for Arp2/3 in nontypeable Haemophilus influenzae invasion. Direct inhibition of actin branch morphology altered bacterial invasion into host epithelial cells, and is supportive of our efforts to use the information gathered to modify outcomes of disease. The twenty-eight nontypeable Haemophilus influenzae proteins identified participate in carbohydrate and amino acid metabolism, redox homeostasis, and include cell wall-associated metabolic proteins. Quantitative characterization of the molecular signatures of infection will redefine our understanding of host response driven developmental changes during pathogenesis. These data represent the first comprehensive study of host protein and metabolite profiles in vivo in response to infection and show the feasibility of extensive characterization of host protein profiles during disease. Identification of novel protein targets and metabolic biomarkers will advance development of therapeutic and diagnostic options for treatment of disease.

Rationalization of Anomalous Pseudocontact Shifts and Their Solvent Dependence in a Series of C3-Symmetric Lanthanide Complexes.

Bleaney's long-standing theory of magnetic anisotropy has been employed with some success for many decades to explain paramagnetic NMR pseudocontact shifts, and has been the subject of many subsequent approximations. Here, we present a detailed experimental and theoretical investigation accounting for the anomalous solvent dependence of NMR shifts for a series of lanthanide(III) complexes, namely [LnL1] (Ln = Eu, Tb, Dy, Ho, Er, Tm, and Yb; L1: 1,4,7-tris[(6-carboxypyridin-2-yl)methyl]-1,4,7-triazacyclononane), taking into account the effect of subtle ligand flexibility on the electronic structure. We show that the anisotropy of the room temperature magnetic susceptibility tensor, which in turn affects the sign and magnitude of the pseudocontact chemical shift, is extremely sensitive to minimal structural changes in the first coordination sphere of L1. We show that DFT structural optimizations do not give accurate structural models, as assessed by the experimental chemical shifts, and thus we determine a magnetostructural correlation and employ this to evaluate the accurate solution structure for each [LnL1]. This approach allows us to explain the counterintuitive pseudocontact shift behavior, as well as a striking solvent dependence. These results have important consequences for the analysis and design of novel magnetic resonance shift and optical emission probes that are sensitive to the local solution environment and polarity.

PARASHIFT Probes: Solution NMR and X-ray Structural Studies of Macrocyclic Ytterbium and Yttrium Complexes.

Ytterbium and yttrium complexes of octadentate ligands based on 1,4,7,10-tetraazacyclododecane with a coordinated pyridyl group and either tricarboxylate (L1) or triphosphinate (L2) donors form twisted-square-antiprismatic structures. The former crystallizes in the centrosymmetric group P21/c, with the two molecules related by an inversion center, whereas the latter was found as an unusual kryptoracemate in the chiral space group P21. Pure shift NMR and EXSY spectroscopy allowed the dynamic exchange between the (RRR)-Δ-(δδδδ) and (RRR)-Λ-(λλλλ) TSAP diastereomers of the [Y.L2] complex to be detected. The rate-limiting step in the exchange between Δ and Λ isomers involves cooperative ligand arm rotation, which is much faster for [Ln.L1] than for [Ln.L2]. Detailed analysis of NOESY, COSY, HSQC, and HMBC spectra confirms that the major conformer in solution is (RRR)-Λ-(λλλλ), consistent with crystal structure analysis and DFT calculations. The magnetic susceptibility tensors for [Yb.L1] and [Yb.L2], obtained from a full pseudocontact chemical shift analysis, are very different, in agreement with a CASSCF calculation. The remarkably different pseudocontact shift behavior is explained by the change in the orientation of the pseudocontact shift field, as defined by the Euler angles of the susceptibility tensor.

Beyond Bleaney's Theory: Experimental and Theoretical Analysis of Periodic Trends in Lanthanide-Induced Chemical Shift.

A detailed analysis of paramagnetic NMR shifts in a series of isostructural lanthanide complexes relavant to PARASHIFT contrast agents reveals unexpected trends in the magnetic susceptibility anisotropy that cannot be explained by the commonly used Bleaney's theory. Ab initio calculations reveal that the primary assumption of Bleaney's theory-that thermal energy is larger than the ligand field splitting-does not hold for the lanthanide complexes in question, and likely for a large fraction of lanthanide complexes in general. This makes the orientation of the magnetic susceptibility tensor differ significantly between complexes of different lanthanides with the same ligand: one of the most popular assumptions about isostructural lanthanide series is wrong.

Association of O-Antigen Serotype with the Magnitude of Initial Systemic Cytokine Responses and Persistence in the Urinary Tract.

UNLABELLED: Urinary tract infection (UTI) is one of the most common ailments requiring both short-term and prophylactic antibiotic therapies. Progression of infection from the bladder to the kidney is associated with more severe clinical symptoms (e.g., fever and vomiting) as well as with dangerous disease sequelae (e.g., renal scaring and sepsis). Host-pathogen interactions that promote bacterial ascent to the kidney are not completely understood. Prior studies indicate that the magnitude of proinflammatory cytokine elicitation in vitro by clinical isolates of uropathogenic Escherichia coli (UPEC) inversely correlates with the severity of clinical disease. Therefore, we hypothesize that the magnitude of initial proinflammatory responses during infection defines the course and severity of disease. Clinical UPEC isolates obtained from patients with a nonfebrile UTI elicited high systemic proinflammatory responses early during experimental UTI in a murine model and were attenuated in bladder and kidney persistence. Conversely, UPEC isolates obtained from patients with febrile UTI elicited low systemic proinflammatory responses early during experimental UTI and exhibited prolonged persistence in the bladder and kidney. Soluble factors in the supernatant from saturated cultures as well as the lipopolysaccharide (LPS) serotype correlated with the magnitude of proinflammatory responses in vitro. Our data suggest that the structure of the O-antigen sugar moiety of the LPS may determine the strength of cytokine induction by epithelial cells. Moreover, the course and severity of disease appear to be the consequence of the magnitude of initial cytokines produced by the bladder epithelium during infection. IMPORTANCE: The specific host-pathogen interactions that determine the extent and course of disease are not completely understood. Our studies demonstrate that modest changes in the magnitude of cytokine production observed using in vitro models of infection translate into significant ramifications for bacterial persistence and disease severity. While many studies have demonstrated that modifications of the LPS lipid A moiety modulate the extent of Toll-like receptor 4 (TLR4) activation, our studies implicate the O-antigen sugar moiety as another potential rheostat for the modulation of proinflammatory cytokine production.

Haemophilus responses to nutritional immunity: epigenetic and morphological contribution to biofilm architecture, invasion, persistence and disease severity.

In an effort to suppress microbial outgrowth, the host sequesters essential nutrients in a process termed nutritional immunity. However, inflammatory responses to bacterial insult can restore nutritional resources. Given that nutrient availability modulates virulence factor production and biofilm formation by other bacterial species, we hypothesized that fluctuations in heme-iron availability, particularly at privileged sites, would similarly influence Haemophilus biofilm formation and pathogenesis. Thus, we cultured Haemophilus through sequential heme-iron deplete and heme-iron replete media to determine the effect of transient depletion of internal stores of heme-iron on multiple pathogenic phenotypes. We observed that prior heme-iron restriction potentiates biofilm changes for at least 72 hours that include increased peak height and architectural complexity as compared to biofilms initiated from heme-iron replete bacteria, suggesting a mechanism for epigenetic responses that participate in the changes observed. Additionally, in a co-infection model for human otitis media, heme-iron restricted Haemophilus, although accounting for only 10% of the inoculum (90% heme-iron replete), represented up to 99% of the organisms recovered at 4 days. These data indicate that fluctuations in heme-iron availability promote a survival advantage during disease. Filamentation mediated by a SulA-related ortholog was required for optimal biofilm peak height and persistence during experimental otitis media. Moreover, severity of disease in response to heme-iron restricted Haemophilus was reduced as evidenced by lack of mucosal destruction, decreased erythema, hemorrhagic foci and vasodilatation. Transient restriction of heme-iron also promoted productive invasion events leading to the development of intracellular bacterial communities. Taken together, these data suggest that nutritional immunity, may, in fact, foster long-term phenotypic changes that better equip bacteria for survival at infectious sites.

Otitis media: Interactions between host and environment, immune and inflammatory responses.

OBJECTIVE: To review and highlight progress in otitis media (OM) research in the areas of immunology, inflammation, environmental influences and host-pathogen responses from 2019 to 2023. Opportunities for innovative future research were also identified. DATA SOURCES: PubMed database of the National Library of Medicine. REVIEW METHODS: Key topics were assigned to each panel member for detailed review. Search of the literature was from June 2019 until February 2023. Draft reviews were collated, circulated, and discussed among panel members at the 22nd International Symposium on Recent Advances in Otitis Media in June 2023. The final manuscript was prepared and approved by all the panel members. CONCLUSIONS: Important advances were identified in: environmental influences that enhance OM susceptibility; polymicrobial middle ear (ME) infections; the role of adaptive immunity defects in otitis-proneness; additional genes linked to OM; leukocyte contributions to OM pathogenesis and recovery; and novel interventions in OM based on host responses to infection. Innovative areas of research included: identification of novel bacterial genes and pathways important for OM persistence, bacterial adaptations and evolution that enhance chronicity; animal and human ME gene expression, including at the single-cell level; and Sars-CoV-2 infection of the ME and Eustachian tube.

The Haemophilus influenzae Sap transporter mediates bacterium-epithelial cell homeostasis.

Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharynx and a causative agent of otitis media and other diseases of the upper and lower human airway. During colonization within the host, NTHI must acquire essential nutrients and evade immune attack. We previously demonstrated that the NTHI Sap transporter, an inner membrane protein complex, mediates resistance to antimicrobial peptides and is required for heme homeostasis. We hypothesized that Sap transporter functions are critical for NTHI interaction with the host epithelium and establishment of colonization. Thus, we cocultured the parent or the sapA mutant on polarized epithelial cells grown at an air-liquid interface, as a physiological model of NTHI colonization, to determine the contribution of the Sap transporter to bacterium-host cell interactions. Although SapA-deficient NTHI was less adherent to epithelial cells, we observed a significant increase in invasive bacteria compared to the parent strain. Upon internalization, the sapA mutant appeared free in the cytoplasm, whereas the parent strain was primarily found in endosomes, indicating differential subcellular trafficking. Additionally, we observed reduced inflammatory cytokine production by the epithelium in response to the sapA mutant strain compared to the parental strain. Furthermore, chinchilla middle ears challenged with the sapA mutant demonstrated a decrease in disease severity compared to ears challenged with the parental strain. Collectively, our data suggest that NTHI senses host environmental cues via Sap transporter function to mediate interaction with host epithelial cells. Epithelial cell invasion and modulation of host inflammatory cytokine responses may promote NTHI colonization and access to essential nutrients.

Ferric uptake regulator and its role in the pathogenesis of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is a commensal microorganism of the human nasopharynx, and yet is also an opportunistic pathogen of the upper and lower respiratory tracts. Host microenvironments influence gene expression patterns, likely critical for NTHi persistence. The host sequesters iron as a mechanism to control microbial growth, and yet iron limitation influences gene expression and subsequent production of proteins involved in iron homeostasis. Careful regulation of iron uptake, via the ferric uptake regulator Fur, is essential in multiple bacteria, including NTHi. We hypothesized therefore that Fur contributes to iron homeostasis in NTHi, is critical for bacterial persistence, and likely regulates expression of virulence factors. Toward this end, fur was deleted in the prototypic NTHi clinical isolate, 86-028NP, and we assessed gene expression regulated by Fur. As expected, expression of the majority of genes that encode proteins with predicted roles in iron utilization was repressed by Fur. However, 14 Fur-regulated genes encode proteins with no known function, and yet may contribute to iron utilization or other biological functions. In a mammalian model of human otitis media, we determined that Fur was critical for bacterial persistence, indicating an important role for Fur-mediated iron homeostasis in disease progression. These data provide a profile of genes regulated by Fur in NTHi and likely identify additional regulatory pathways involved in iron utilization. Identification of such pathways will increase our understanding of how this pathogen can persist within host microenvironments, as a common commensal and, importantly, as a pathogen with significant clinical impact.

Sap transporter mediated import and subsequent degradation of antimicrobial peptides in Haemophilus.

Antimicrobial peptides (AMPs) contribute to host innate immune defense and are a critical component to control bacterial infection. Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharyngeal mucosa, yet is commonly associated with opportunistic infections of the upper and lower respiratory tracts. An important aspect of NTHI virulence is the ability to avert bactericidal effects of host-derived antimicrobial peptides (AMPs). The Sap (sensitivity to antimicrobial peptides) ABC transporter equips NTHI to resist AMPs, although the mechanism of this resistance has remained undefined. We previously determined that the periplasmic binding protein SapA bound AMPs and was required for NTHI virulence in vivo. We now demonstrate, by antibody-mediated neutralization of AMP in vivo, that SapA functions to directly counter AMP lethality during NTHI infection. We hypothesized that SapA would deliver AMPs to the Sap inner membrane complex for transport into the bacterial cytoplasm. We observed that AMPs localize to the bacterial cytoplasm of the parental NTHI strain and were susceptible to cytoplasmic peptidase activity. In striking contrast, AMPs accumulated in the periplasm of bacteria lacking a functional Sap permease complex. These data support a mechanism of Sap mediated import of AMPs, a novel strategy to reduce periplasmic and inner membrane accumulation of these host defense peptides.

Determination of molecular hydration in solution via changes in magnetic anisotropy.

The hydration behaviour of coordination complexes is important for understanding their roles as bio-imaging agents. Determination of hydration is difficult, and various optical and NMR-based techniques have been used. Here we use EPR spectroscopy to unambiguously demonstrate that a t-butyl-pyridyl-functionalised ErIII DOTA derivative coordinates water, while its methylphosphinate analogue does not.

The use of Escherichia coli strain Nissle 1917 shows promise for improving gastrointestinal and urinary health in dogs.

OBJECTIVES: To investigate the probiotic Escherichia coli Nissle 1917 (EcN) in canine idiopathic diarrhea and urinary tract infections. ANIMALS/SAMPLES: The utility of EcN was explored in a 3-phase study from March 2017 to June 2020. Eighty-nine dogs with idiopathic diarrhea were included in phase 1, 3 healthy dogs were included in phase 2, and uropathogenic E coli (UPEC) isolates from 38 dogs with urinary tract infections were included in phase 3. PROCEDURES: In phase 1, dogs with diarrhea were prospectively enrolled in a randomized study to receive EcN (108 EcN bacteria/mL; < 10 kg received 5 mL/dose, 10 to 25 kg received 10 mL/dose, or > 25 kg received 15 mL/dose) or placebo for 3 days, followed by a 15-day observation phase. In phase 2, healthy dogs received EcN as described in phase 1, with feces analyzed for E coli populations and microbiome composition at days 0, 3, and 7. In phase 3, EcN efficacy was tested by in vitro plate assay against UPEC isolates. RESULTS: Median duration of abnormal stool consistency, time to response, and duration of diarrhea were shorter for dogs that received EcN (5.0, 3.0, and 2.0 days, respectively) versus the placebo (7.0, 5.0, and 4.0 days, respectively) (P = .21, P = .05, and P = .039, respectively). EcN induced shifts in E coli diversity in healthy dogs while having minimal impact on overall microbiome structure. Furthermore, 68% of the canine UPEC isolates were susceptible to EcN in vitro. CLINICAL RELEVANCE: EcN improved the treatment of idiopathic diarrhea, colonized the gastrointestinal tract during the trial, and displayed in vitro competition with UPEC.