Persistence of Bartonella spp. stealth pathogens: from subclinical infections to vasoproliferative tumor formation

Bartonella spp. are facultative intracellular bacteria that typically cause a long-lasting intraerythrocytic bacteremia in their mammalian reservoir hosts, thereby favoring transmission by blood-sucking arthropods. In most cases, natural reservoir host infections are subclinical and the relapsing intraerythrocytic bacteremia may last weeks, months, or even years. In this review, we will follow the infection cycle of Bartonella spp. in a reservoir host, which typically starts with an intradermal inoculation of bacteria that are superficially scratched into the skin from arthropod feces and terminates with the pathogen exit by the blood-sucking arthropod. The current knowledge of bacterial countermeasures against mammalian immune response will be presented for each critical step of the pathogenesis. The prevailing models of the still-enigmatic primary niche and the anatomical location where bacteria reside, persist, and are periodically seeded into the bloodstream to cause the typical relapsing Bartonella spp. bacteremia will also be critically discussed. The review will end up with a discussion of the ability of Bartonella spp., namely Bartonella henselae, Bartonella quintana, and Bartonella bacilliformis, to induce tumor-like vascular deformations in humans having compromised immune response such as in patients with AID

Exotoxin-Targeted Drug Modalities as Antibiotic Alternatives

The paradigm of antivirulence therapy dictates that bacterial pathogens are specifically disarmed but not killed by neutralizing their virulence factors. Clearance of the invading pathogen by the immune system is promoted. As compared to antibiotics, the pathogen-selective antivirulence drugs hold promise to minimize collateral damage to the beneficial microbiome. Also, selective pressure for resistance is expected to be lower because bacterial viability is not directly affected. Antivirulence drugs are being developed for stand-alone prophylactic and therapeutic treatments but also for combinatorial use with antibiotics. This Review focuses on drug modalities that target bacterial exotoxins after the secretion or release-upon-lysis. Exotoxins have a significant and sometimes the primary role as the disease-causing virulence factor, and thereby they are attractive targets for drug development. We describe the key pre-clinical and clinical trial data that have led to the approval of currently used exotoxin-targeted drugs, namely the monoclonal antibodies bezlotoxumab (toxin B/TcdB, Clostridioides difficile), raxibacumab (anthrax toxin, Bacillus anthracis), and obiltoxaximab (anthrax toxin, Bacillus anthracis), but also to challenges with some of the promising leads. We also highlight the recent developments in pre-clinical research sector to develop exotoxin-targeted drug modalities, i.e., monoclonal antibodies, antibody fragments, antibody mimetics, receptor analogs, neutralizing scaffolds, dominant-negative mutants, and small molecules. We describe how these exotoxin-targeted drug modalities work with high-resolution structural knowledge and highlight their advantages and disadvantages as antibiotic alternatives

Host poly(ADP-ribose) polymerases (PARPs) in acute and chronic bacterial infections

AbstractProtein ADP-ribosylation is a reversible post-translational modification, which alters protein activity, localization, interactome or stability, leading to perturbation of cell signaling. This review summarizes the emerging data indicating that host cell ADP-ribosylating enzymes, poly(ADP-ribose) polymerases (PARPs), influence the course of a bacterial infection, in parallel to ADP-ribosylating bacterial toxins. Host cell PARP targeting could be an efficient therapeutic approach to treat certain bacterial infections, possibly by repurposing the approved or clinical trial PARP inhibitors developed for cancer therapy.</div

Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin

Bordetella pertussis causes the severe childhood disease whooping cough, by releasing several toxins, including pertussis toxin (PT) as a major virulence factor. PT is an AB(5)-type toxin, and consists of the enzymatic A-subunit PTS1 and five B-subunits, which facilitate binding to cells and transport of PTS1 into the cytosol. PTS1 ADP-ribosylates alpha-subunits of inhibitory G-proteins (G alpha i) in the cytosol, which leads to disturbed cAMP signaling. Since PT is crucial for causing severe courses of disease, our aim is to identify new inhibitors against PT, to provide starting points for novel therapeutic approaches. Here, we investigated the effect of human antimicrobial peptides of the defensin family on PT. We demonstrated that PTS1 enzyme activity in vitro was inhibited by alpha-defensin-1 and -5, but not beta-defensin-1. The amount of ADP-ribosylated G alpha i was significantly reduced in PT-treated cells, in the presence of alpha-defensin-1 and -5. Moreover, both alpha-defensins decreased PT-mediated effects on cAMP signaling in the living cell-based interference in the G alpha i-mediated signal transduction (iGIST) assay. Taken together, we identified the human peptides alpha-defensin-1 and -5 as inhibitors of PT activity, suggesting that these human peptides bear potential for developing novel therapeutic strategies against whooping cough

IGIST - A Kinetic Bioassay for Pertussis Toxin Based on Its Effect on Inhibitory GPCR Signaling

Detection of pertussis toxin (PTX) activity is instrumental for the development and manufacturing of pertussis vaccines. These quality and safety measures require thousands of mice annually. Here, we describe Interference in Gαi-mediated Signal Transduction (iGIST), an animal-free kinetic bioassay for detection of PTX, by measuring its effect on inhibitory G protein-coupled receptor (GPCR) signaling. PTX ADP-ribosylates inhibitory α-subunits of the heterotrimeric G proteins, thereby perturbing the inhibitory GPCR signaling. iGIST is based on HEK293 cells coexpressing a somatostatin receptor 2 (SSTR2), which is an inhibitory GPCR controllable by a high-affinity agonist octreotide; and a luminescent 3′5′-cyclic adenosine monophosphate (cAMP) probe. iGIST has a low sensitivity threshold in the pg/mL range of PTX, surpassing by 100-fold in a parallel analysis the currently used in vitro end-point technique to detect PTX, the cluster formation assay (CFA) in Chinese hamster ovary cells. iGIST also detects PTX in complex samples, i.e., a commercial PTX-toxoid-containing pertussis vaccine that was spiked with an active PTX. iGIST has an objective digital readout and is observer independent, offering prospects for automation. iGIST emerges as a promising animal-free alternative to detect PTX activity in the development and manufacturing of pertussis vaccines. iGIST is also expected to facilitate basic PTX research, including identification and characterization of novel compounds interfering with PTX.</p

Crystal structures of pertussis toxin with NAD(+) and analogs provide structural insights into the mechanism of its cytosolic ADP-ribosylation activity

Bordetella pertussis is the causative agent of whooping cough, a highly contagious respiratory disease. Pertussis toxin (PT), a major virulence factor secreted by B. pertussis, is an AB5-type protein complex topologically related to cholera toxin. The PT protein complex is internalized by host cells and follows a retrograde trafficking route to the endoplasmic reticulum, where it subsequently dissociates. The released enzymatic S1 subunit is then translocated from the endoplasmic reticulum into the cytosol and subsequently ADP-ribosylates the inhibitory alpha-subunits (G alpha i) of heterotrimeric G proteins, thus promoting dysregulation of G protein-coupled receptor signaling. However, the mechanistic details of the ADP-ribosylation activity of PT are not well understood. Here, we describe crystal structures of the S1 subunit in complex with nicotinamide adenine dinucleotide (NAD+), with NAD+ hydrolysis products ADP-ribose and nicotinamide, with NAD+ analog PJ34, and with a novel NAD+ analog formed upon S1 subunit crystallization with 3-amino benzamide and NAD+, which we name benzamide amino adenine dinucleotide. These crystal structures provide unprecedented insights into pre-and post-NAD+ hydrolysis steps of the ADP-ribosyltransferase activity of PT. We propose that these data may aid in rational drug design approaches and further development of PT-specific small-molecule inhibitors

Crystal structures of pertussis toxin with NAD+ and analogs provide structural insights into the mechanism of its cytosolic ADP-ribosylation activity

Bordetella pertussis is the causative agent of whooping cough, a highly contagious respiratory disease. Pertussis toxin (PT), a major virulence factor secreted by B. pertussis, is an AB5-type protein complex topologically related to cholera toxin. The PT protein complex is internalized by host cells and follows a retrograde trafficking route to the endoplasmic reticulum (ER), where it subsequently dissociates. The released enzymatic S1 subunit is then translocated from the ER into the cytosol and subsequently ADP-ribosylates the inhibitory alpha-subunits (Gαi) of heterotrimeric G proteins, thus promoting dysregulation of G-protein coupled receptor (GPCR) signaling. However, the mechanistic details of the ADP-ribosylation activity of PT are not well understood. Here, we describe crystal structures of the S1 subunit in complex with nicotinamide adenine dinucleotide (NAD+), with NAD+ hydrolysis products ADP-ribose and nicotinamide, with NAD+ analog PJ34, and with a novel NAD+ analog formed upon S1 subunit crystallization with 3-amino benzamide (3AB) and NAD+, which we name benzamide amino adenine dinucleotide (BaAD). These crystal structures provide unprecedented insights into pre- and post-NAD+ hydrolysis steps of the ADP-ribosyltransferase activity of PT. We propose that these data may aid in rational drug design approaches and further development of PT-specific small molecule inhibitors

Microbiome Analysis Reveals the Presence of Bartonella spp. and Acinetobacter spp. in Deer Keds (Lipoptena cervi)

The deer ked (Lipoptena cervi) is distributed in Europe, North America, and Siberia and mainly infests cervids as roe deer, fallow deer, and moose. From a one health perspective, deer keds occasionally bite other animals or humans and are a potential vector for Bartonella schoenbuchensis. This bacterium belongs to a lineage of ruminant-associated Bartonella spp. and is suspected to cause dermatitis and febrile diseases in humans. In this study, we analyzed the microbiome from 130 deer keds collected from roe deer, fallow deer and humans in the federal states of Hesse, Baden-Wuerttemberg, and Brandenburg, Germany. Endosymbiontic Arsenophonus spp. and Bartonella spp. represented the biggest portion (~90%) of the microbiome. Most Bartonella spp. (n = 93) were confirmed to represent B. schoenbuchensis. In deer keds collected from humans, no Bartonella spp. were detected. Furthermore, Acinetobacter spp. were present in four samples, one of those was confirmed to represent A. baumannii. These data suggest that deer keds harbor only a very narrow spectrum of bacteria which are potentially pathogenic for animals of humans

Combination of microbiome analysis and serodiagnostics to assess the risk of pathogen transmission by ticks to humans and animals in central Germany

BackgroundArthropod-borne diseases remain a major health-threat for humans and animals worldwide. To estimate the distribution of pathogenic agents and especially Bartonella spp., we conducted tick microbiome analysis and determination of the infection status of wild animals, pets and pet owners in the state of Hesse, Germany.ResultsIn total, 189 engorged ticks collected from 163 animals were tested. Selected ticks were analyzed by next generation sequencing (NGS) and confirmatory PCRs, blood specimens of 48 wild animals were analyzed by PCR to confirm pathogen presence and sera of 54 dogs, one cat and 11 dog owners were analyzed by serology. Bartonella spp. were detected in 9.5% of all ticks and in the blood of 17 roe deer. Further data reveal the presence of the human and animal pathogenic species of genera in the family Spirochaetaceae (including Borrelia miyamotoi and Borrelia garinii), Bartonella spp. (mainly Bartonella schoenbuchensis), Rickettsia helvetica, Francisella tularensis and Anaplasma phagocytophilum in ticks. Co-infections with species of several genera were detected in nine ticks. One dog and five dog owners were seropositive for anti-Bartonella henselae-antibodies and one dog had antibodies against Rickettsia conorii.ConclusionsThis study provides a snapshot of pathogens circulating in ticks in central Germany. A broad range of tick-borne pathogens are present in ticks, and especially in wild animals, with possible implications for animal and human health. However, a low incidence of Bartonella spp., especially Bartonella henselae, was detected. The high number of various detected pathogens suggests that ticks might serve as an excellent sentinel to detect and monitor zoonotic human pathogens

Homogeneous Dual-Parametric-Coupled Assay for Simultaneous Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring

We have developed a rapid and sensitive single-well dual-parametric method introduced in linked RAS nucleotide exchange and RAS/RAF-RBD interaction assays. RAS mutations are frequent drivers of multiple different human cancers, but the development of therapeutic strategies has been challenging. Traditionally, efforts to disrupt the RAS function have focused on nucleotide exchange inhibitors, GTP-RAS interaction inhibitors, and activators increasing GTPase activity of mutant RAS proteins. As the amount of biological knowledge grows, targeted biochemical assays enabling high-throughput screening have become increasingly interesting. We have previously introduced a homogeneous quenching resonance energy transfer (QRET) assay for nucleotide binding studies with RAS and heterotrimeric G proteins. Here, we introduce a novel homogeneous signaling technique called QTR-FRET, which combine QRET technology and time-resolved Förster resonance energy transfer (TR-FRET). The dual-parametric QTR-FRET technique enables the linking of guanine nucleotide exchange factor-induced Eu3+-GTP association to RAS, monitored at 615 nm, and subsequent Eu3+-GTP-loaded RAS interaction with RAF-RBD-Alexa680 monitored at 730 nm. Both reactions were monitored in a single-well assay applicable for inhibitor screening and real-time reaction monitoring. This homogeneous assay enables separable detection of both nucleotide exchange and RAS/RAF interaction inhibitors using low nanomolar protein concentrations. To demonstrate a wider applicability as a screening and real-time reaction monitoring method, the QTR-FRET technique was also applied for G(i)α GTP-loading and pertussis toxin-catalyzed ADP-ribosylation of G(i)α, for which we synthesized a novel γ-GTP-Eu3+ molecule. The study indicates that the QTR-FRET detection technique presented here can be readily applied to dual-parametric assays for various targets